Diagnostic detection device and method

ABSTRACT

The invention provides an improved test cell for detecting the presence of an analyte in a liquid sample. The device has an elongate casing defining a liquid sample inlet, a reservoir volume, a test volume, and a window through the casing at the test volume. Disposed within the cell is a sample absorbent, a novel biphasic substrate and a reservoir, together capable of transporting an aqueous solution within the casing along a flow path extending from the sample inlet through the test volume and into the reservoir volume. The invention further comprises a method for detecting the presence of an analyte in a liquid sample using the device and a biphasic chromatographic material for carrying out the method.

This application is a continuation of U.S. application Ser. No.09/951,007 filed Sep. 12, 2001, now U.S. Pat. No. 6,767,714, which is adivisional of U.S. application Ser. No. 08/432,894, filed May 2, 1995,now U.S. Pat. No. 6,319,676, the disclosures of each of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention relates to assays for an analyte, such as anantigen, in a liquid sample, such as body fluid. More particularly, thepresent invention relates to a method and device for the detection of ananalyte in a body fluid using a lateral flow test cell containing anovel biphasic chromatographic substrate.

Many types of ligand-receptor assays have been used to detect thepresence of various substances in body fluids such as urine or blood.These assays typically involve antigen-antibody reactions, syntheticconjugates comprising enzymatic, fluorescent, or visually observabletags, and specially designed reactor chambers. In most of these assays,there is a receptor (e.g. an antibody) which is specific for theselected antigen, and a means for detecting the presence and/or amountof the antigen-antibody reaction product. Most current tests aredesigned to make a quantitative determination, but in many circumstancesall that is required is a positive/negative indication. Examples of suchqualitative assays include blood typing, pregnancy testing and manytypes of urinalysis. For these tests, visually observable indicia suchas the presence of agglutination or a color change are preferred.

The positive/negative assays must be very sensitive because of the oftensmall concentration of the ligand of interest in the test fluid. Falsepositives can be troublesome, particularly with agglutination and otherrapid detection methods such as dipstick and color change tests. Becauseof these problems, sandwich assays and other sensitive detection methodswhich use metal sols or other types of colored particles have beendeveloped. These techniques have not solved all of the problemsencountered in these rapid detection methods, however. It is an objectof the present invention to provide an improved detection device andmethod having greater sensitivity and discrimination for analytes ofinterest. Another object of the invention is to provide an assay devicewhich is simpler to manufacture.

SUMMARY OF THE INVENTION

The present invention provides a rapid, sensitive device and method fordetecting the presence of analytes in body fluids. The method and devicehave high sensitivity and result in virtually no false positives. Use ofthe present device and method provides an assay system which involves aminimal number of procedural steps, and reproducibly yields reliableresults even when used by untrained persons.

The device and method utilize a unique biphasic chromatographic mediumwhich enhances the speed and sensitivity of the assay. According to thepresent invention, a biphasic substrate element is provided comprising arelease medium joined to a capture medium located downstream of saidrelease medium. The release and capture media preferably comprise twodifferent materials or phases having different specific characteristics.The two phases are joined together to form a single liquid path suchthat a solvent front can travel unimpeded from the proximal (upstream)end of the release medium to the distal (downstream) end of the capturemedium.

The release medium comprises a bibulous, hydrophilic material, such asabsorbent paper. Preferred materials for use as a release medium includecotton linter paper, cellulosic paper, or paper made of cellulosetogether with a polymeric fibrous material, such as polyamide or rayonfibers, and glass fiber material. The primary function of the releasemedium is first to support and to subsequently release and transportvarious immunological components of the assay, such as a labeled bindingmember and a capturable component, both of which have specific affinityfor the analyte of interest. This release and transport occurs duringroutine operation of the assay.

The capture medium comprises a hydrophilic polymeric material,preferably a nitrocellulose or nylon membrane. The preferred materialsfor use as a capture medium are microporous films or membranes whichpermit protein reagents to be immobilized directly on the membrane bypassive adsorption without need for chemical or physical fixation. Forthis purpose, membranes of nitrocellulose, nylon 66 or similar materialsare preferred most preferably having a pore size in the range of fromabout 5μ to about 20μ. The nitrocellulose membrane may be nitrocellulosealone or a mixed ester of nitrocellulose. The nitrocellulose membranepreferably is coated or laminated onto a translucent or transparentpolymeric film to provide physical support for the membrane. In acurrently preferred embodiment, a nitrocellulose polymer which has beencast onto a polyester film such as Mylar® is used. Alternatively, anitrocellulose membrane laminated onto a polyester film also may beused. Other backing materials besides polyester may be used. The primaryfunction of the capture medium is to immobilize an immunological orchemical affinity agent at one or more capture sites for capturing thereagents released from the release medium.

As stated above, the release and capture media are joined together toform a single liquid path. Reagents for detecting labeling and capturingthe analyte of interest are disposed on the release and capture media.Located on the release medium is a binding member reactive with a firstepitope of the analyte of interest. The binding member is labeled with adetectable marker. A capturable component is located on the releasemedium downstream of the binding member, which component comprises abinding agent reactive with a second epitope of the analyte and onemember of an affinity pair. The capturable component is capable offorming a complex with the labeled binding member and the analyte. Thelabeled binding member and the capturable component both are releasablybound to the release medium such that when the solvent front created bythe liquid sample being analyzed passes through the release medium, thelabeled binding member and the capturable component both becomesolubilized by the liquid and flow with the solvent along the liquidpath. In operation, if any analyte is present in the liquid sample, itreacts first with the labeled binding member, then with the capturablecomponent as the front advances along the liquid path. By the time thesolvent front reaches the capture medium section of the biphasicmaterial, the capturable complex has formed.

The capture site located on the capture medium comprises the othermember of the affinity pair specific for the capturable component. Theaffinity member is immobilized, preferably by simple adsorption, at thecapture site, and does not advance with the solvent front.

In a preferred embodiment, a control site also is located on the capturemedium downstream of the capture site. The control site has immobilizedthereon a binding agent having an affinity for the labeled bindingmember. The binding agent will capture any labeled binding member whichis not captured at the upstream capture site. In operation, the presenceof the detectable marker at the control site indicates that sorptivetransport has operated properly.

The present invention further provides a device for detecting thepresence of an analyte in a liquid sample. The device comprises anelongate casing housing the biphasic medium, and defining a liquidsample inlet, a reservoir volume, a test volume interposed between theinlet and reservoir volume, and a window through the casing to observethe test result. Preferably, the sample inlet and the window are locatedin opposite sides of the casing. The casing is adapted to receive theassay materials, which are disposed on the biphasic medium sequentiallywithin the casing. The assay materials comprise an optional sampleabsorbent, the biphasic chromatographic substrate and a reservoirabsorbent. The chromatographic medium is positioned within the casingsuch that the capture site, and the control site if applicable, arevisible through the window. The sample absorbent, biphasicchromatographic substrate and reservoir absorbent are in fluidcommunication and together form a liquid path.

In a currently preferred embodiment, the device comprises a casingdefining a sample inlet, a test volume and reservoir volume. Disposedwithin the casing are a sample absorbent, the biphasic chromatographicsubstrate and reservoir absorbent. The sample absorbent is disposedwithin the casing opposite the sample inlet. Located downstream of thesample absorbent is the biphasic chromatographic substrate comprising arelease medium and a capture medium joined together to form a singleliquid path. The release medium preferably comprises sorbent paper, andthe capture medium preferably comprises nitrocellulose membrane. Therelease and capture media preferably are both laminated onto atransparent plastic film or sheet. Disposed on said release medium is(i) a binding member comprising a specific binding protein, e.g., amonoclonal antibody reactive with a first epitope of said analyte, saidantibody being labeled with a visually detectable marker such ascolloidal gold particles; and (ii) a capturable component comprising abiotinylated binding protein, e.g., an antibody preferably disposeddownstream of said labeled antibody. The biotinylated antibody isreactive with a second epitope of the analyte and is capable of forminga complex with the labeled antibody and the analyte. Disposed on thecapture medium is a capture site for capturing and immobilizing thecomplex. The capture site has immobilized thereon a capture componentwhich has a high affinity for the biotin portion of the complex,preferably streptavidin.

The biphasic chromatographic medium preferably further comprises acontrol site disposed on the capture medium downstream of said capturesite. The control site has immobilized thereon an agent capable ofcapturing said labeled antibody. The primary function of the controlsite is to capture and immobilize labeled antibody which has not beencaptured at the capture site. In the currently preferred embodiment, thecontrol site has immobilized thereon polyclonal antisera specific forthe labeled antibody. The appearance of color from the gold particles atthe control site indicates proper functioning of the test, irrespectiveof the presence or absence of analyte in the sample. Both the captureand control sites must be visible through the window of the casing.

In the method of the invention, the proximal end of the biphasicsubstrate is contacted with the liquid sample being analyzed. The liquidsample travels impelled by surface effects such as by capillary actionalong the liquid path formed by the substrate. If the analyte ofinterest is present in the sample, it sequentially reacts with thelabeled binding member and the capturable component, forming thecapturable complex, followed by reaction of the complex with theimmobilized capture component at the capture site. This process resultsin the labeled complex accumulating at the capture site. The presence ofthe analyte is determined by observing the presence of the detectablemarker at the capture site. If no analyte is present in the sample, thecapturable complex does not form and no detectable marker will bepresent at the capture site. If a control site is present, the unboundcomplex or the free labeled binding member will accumulate at thecontrol site.

The method of the invention also may be designed to exploit conventional“sandwich” or “competitive” techniques. In the case of the sandwichtechnique, the labeled binding member comprises an antibody which bindsto an epitope on the analyte of interest to form a labeledantibody-antigen complex. This complex then migrates to the capture siteto react with a capturable component which, in this embodiment,comprises a second antibody specific for a second epitope of saidanalyte. For example, in the case of biotin, the affinity member may bestreptavidin. At the capture site, the analyte and labeled antibodyreacts with the immobilized capture member to form a “sandwich” of thesecond antibody, analyte and labeled antibody. This sandwich complex isprogressively produced at the capture site as sample continuously passesby. As more and more labeled conjugate is immobilized at the capturesite, the colored particles aggregate and become visible through thewindow of the casing, indicating the presence of the analyte in theliquid sample. Both in the presence or absence of a detectable level ofanalyte, the colored particles gather at the control site which also isvisible through the window.

In the case of the competitive technique, a known amount of the analyteof interest is present on the release medium disposed upstream of anantibody specific for it. The analyte present in the release medium islabeled. The labeled analyte on the release medium may comprise, forexample, an authentic sample of the analyte, or a fraction thereof whichhas comparable affinity for the antibody. As the liquid sample istransported along the release medium, the labeled analyte present on therelease medium and any unlabeled analyte present in the sample competefor sites of attachment to the antibody. If no analyte is present in thesample, labeled analyte-antibody aggregates at the capture site, and thepresence of color indicates the absence of detectable levels of analytein the sample. If analyte is present, the amount of labeled analytewhich binds at the test site is reduced because of binding of analyte inthe sample with the antibody, and no color, or a paler color, develops.

Alternatively, the system described for “sandwich” assay may be used.The antibody specific for the analyte is biotinylated, with steptavidinbeing immobilized at the capture site.

The use of the colored particle detection system in combination with theunique biphasic substrate enables construction of a family of extremelysensitive assay systems which minimize the occurrence of false positivesand which can be used effectively by untrained persons.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will now be more particularly described withreference to and as illustrated in, but in no manner limited to, theaccompanying drawings, in which:

FIG. 1A is a top view of an embodiment of a test cell useful in thedevice and process of the present invention showing the indicatorwindow;

FIG. 1B is a longitudinal side view of the device of FIG. 1A;

FIG. 1C is a bottom view of the device of FIG. 1A;

FIG. 1D is a tail end view of the device of FIG. 1A;

FIG. 1E is a perspective view of a currently preferred deviceconstructed in accordance with the present invention;

FIG. 2 is a schematic top view of the test device formed by the sampleabsorbent, biphasic substrate and reservoir material;

FIG. 3 is a schematic top view of a biphasic substrate of the presentinvention;

FIG. 4 is a schematic side view of the test device of FIG. 2;

FIG. 5 is a schematic top view of the test device constructed inaccordance with the invention;

FIG. 6 is a schematic top view of a test substrate for use in a dipstickembodiment of the present invention;

FIG. 7 is a perspective view of a dipstick embodiment of a test celluseful in the device and process of the present invention.

In the drawings, like reference characters in the respective drawnFigures indicate corresponding parts.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention involves the use of a novel biphasicchromatographic substrate to achieve an easily readable, sensitive,reproducible indication of the presence of an analyte, such as humanchorionic gonadotropin (hCG), or luteinizing hormone (LH), in a testsample such as, for example, a human urine sample. The method and devicealso may be used to detect the presence of infectious agents in blood,plasma, mucus or other body fluid.

The biphasic chromatographic substrate of the present invention formsthe basis for immunologically based diagnostic tests for the detectionof various analytes. Use of the substrate in a diagnostic device enablesthe operator to determine with high accuracy and sensitivity thepresence or absence of a biological marker which is indicative of aphysiological condition or state.

The biphasic chromatographic substrate involves the union of twodifferent media, each with a specific function. The release medium hasdisposed thereon two dry, diffusible reagents: a binding member specificto a particular site on the analyte labeled with colloidal gold or otherdirect label, and a capturable component comprising a binding memberspecific for a different site on the analyte conjugated to one member ofan affinity pair. Upon reconstitution when in contact with the testsolution, and in the presence of the analyte, the diffusible reagentsreact with the analyte to form a diffusible sandwich which istransported by capillary action to the capture medium. The capturemedium contains two dry, non-diffusible reagents: a capture componentcomprising the other member of the affinity pair and a reagent specificfor the labeled binding member. Upon diffusion into the capture medium,the diffusible sandwich becomes concentrated by the interaction of thecapture affinity member with the capturable affinity moiety yielding avisual signal.

The biphasic chromatographic substrate comprises a release medium joinedto a capture medium in such a way as to form a single liquid path. Therelease medium is formed from a substance which allows for release ofindicator reagents, and the capture medium is formed from a substancewhich permits immobilization of reagents for detection. The releasemedium preferably is composed of a hydrophilic bibulous material, itsprimary function being to hold, release and transport variousimmunological parts of the test such as the labeled test component. Thisrelease and transport occurs during the routine operation of the testingprocedure. Materials useful in forming the release medium include, forexample, cotton linter paper such as S&S 903 and S&S GB002 (availablefrom Schleicher and Schuell, Inc., Keene, N.H.), and BFC 180 (availablefrom Whatman, Fairfield, N.J.), and cellulosic materials such as Grade939 made of cellulose with polyamide and Grade 1281 made of celluloseand rayon with polyamide (available from Filtertek, Inc.) and glassfiber such as Lydall borosilicate (available from Lydall, Inc.,Rochester, N.H.). The release medium preferably is coated with anaqueous solution containing bovine serum albumin (BSA) and a nonionicsurfactant, such as Triton X-100 (available from Rohm & Haas Co.,Philadelphia, Pa.) in order to prevent nonspecific binding andfacilitate release of the diffusible reagents. A combination of about 3%BSA and about 0.1% Triton X-100 is useful for this purpose.

The capture medium preferably comprises a microporous polymeric film ofnitrocellulose, nylon 66, a combination of the two, or various othermaterials of similar nature which are known by those skilled in the art.Pore size preferably is in the range of from about 5μ to about 20μ. Theprimary function of the capture medium is to immobilize thenon-diffusible reagents used to detect the presence of the analyte inthe test. Protein reagents can be immobilized on the capture medium byadsorption, without the need for chemical or physical modifications. Thenitrocellulose may comprise nitrocellulose alone or combined with anester of nitric acid and/or other acids. In a preferred embodiment, thenitrocellulose is directly cast onto a clear polymer film. Commerciallyavailable polyester films such as those available under the tradenameMylar® are useful for this purpose (Mylar® is a trademark of the DuPontDeNemours Company). Nitrocellulose membrane may be fabricated byart-recognized techniques, including direct casting of thenitrocellulose polymer onto a polyester sheet, or by laminating anitrocellulose film with a polyester sheet. Prelaminated or precastsheets useful in the present invention are commercially available, forexample, from Millipore Corporation, Bedford, Mass. and Corning Costar,Norristown, Pa. Both media are in the form of planar strips, which arejoined together to form a single flow path. In a preferred embodiment,the release medium and capture medium are joined by overlapping thedownstream edge of the release medium over the upstream edge of thecapture medium, then adhering the resulting biphasic material to a clearpolymer film or sheet, thereby holding the media in place.

The method for manufacturing the unique biphasic chromatographic mediumused in the present invention is described in detail in U.S. Pat. No.5,846,835, which claims priority to U.S. application Ser. No.08/434,342, the disclosures of each of which are hereby incorporatedherein by reference. Briefly, the release medium and capture medium arepositioned such that they overlap slightly, and an adhesive is disposedon the back of each (the back being the side opposite that which willreceive the reagents). The adhesive may be any pressure sensitive or hotmelt adhesive which does not fill the pores of the release or capturemedium, thereby permitting unimpeded flow of the solvent front throughthe media. Adhesives useful in the present invention are commerciallyavailable, for example, from Adhesives Research Corp. In a currentlypreferred embodiment, the adhesive is disposed on a clear polymerbacking. The overlapped release and capture media then are passedthrough the laminating rollers of a laminating machine together with thebacked adhesive, forming a laminate of the capture and release media,the adhesive and the polymer backing. The resulting laminated biphasicsubstrate then is ready to receive the reagents, which are deposited ascontinuous “stripes” onto the top of the substrate. Once the reagentshave been deposited and dried, if necessary, the substrate is cut intothe desired size.

The diffusible and non-diffusible reagents can be applied to the releaseand capture media, respectively, by any well-known technique. In acurrently preferred embodiment, the diffusible antibody reagents areapplied to the release medium by direct application onto the surface ofthe medium and dried to form a narrow band. The non-diffusible reagentsare applied to the capture medium by passive adsorption.

For use, the biphasic chromatographic substrate is disposed within atest device, which device also forms a part of this invention. Thedevice comprises, at a minimum, a housing encasing the biphasic systemfor conducting the assay. A preferred housing configuration is shown indesign application Ser. No. 29/023,294, now U.S. Pat. No. Des. 361,842,which is incorporated herein by reference. A particularly preferredembodiment of the casing is described in U.S. application Ser. No.08/432,890, now U.S. Pat. No. 5,739,041, which is incorporated herein byreference.

The method and device of the invention cooperate to enable untrainedpersonnel reliably to assay a liquid sample for the presence ofextremely small quantities of a particular analyte, while avoiding falsepositives and simplifying test procedures. The invention is ideal foruse in over-the-counter assay test kits which will enable a consumer toself-diagnose, for example, pregnancy, ovulation, venereal disease andother disease, infection, or clinical abnormality, which results in thepresence of an antigenic marker substance in a body fluid, includingdetermination of the presence of metabolites of drugs or toxins. Theassay process and the device are engineered specifically to detect thepresence of a preselected individual analyte present in a body fluid.

In addition to the biphasic chromatographic substrate the device maycomprise a sample absorbent disposed within the casing proximate thechromatographic substrate and in fluid communication therewith. Thesample absorbent preferably is a bibulous hydrophilic material whichfacilitates absorption and transport of a fluid sample to the biphasicchromatographic medium. Such materials may include cellulose acetate,hydrophilic polyester, other materials having similar properties. Acombination of absorbent materials also may be used. Preferred materialsinclude bonded cellulose acetate, bonded polyolefin or hydrophilicpolyester, such as those materials commercially available from AmericanFiltrona Company (Richmond, Va.). Other preferred materials includeabsorbent papers such as Grade 939 or Grade 1281, available fromFiltertek, Inc. The sample absorbent preferably is coated with abuffered solution containing BSA and a nonionic surfactant, such asTriton X-100. The presence of BSA and surfactant minimize non-specificabsorption of the analyte. A concentration of about 1% BSA and about0.2% surfactant in tris buffer is effective for this purpose.

The device further may comprise a reservoir absorbent disposeddownstream of the chromatographic substrate and in fluid communicationtherewith. By providing a reservoir of sorbent material disposed beyondthe chromatographic substrate, a relatively large volume of the testliquid and any analyte it contains can be drawn through the test area toaid sensitivity. The reservoir material preferably comprises ahydrophilic material which may be the same as the upstream sampleabsorbent. The purpose of the reservoir absorbent is to facilitatecapillary action along the chromatographic substrate and to absorbexcess liquid contained within the device. The reservoir absorbentpreferably compromises absorbent paper made from cotton long linterfibers, such as S&S 300, S&S 470 and S&S 900, (available from Schleicher& Schuell, Inc.) or cellulosic materials, such as Grade 3MM (availablefrom Whatman) and Grade 320 (available from Alhstrom).

Broadly, the device and method of the invention can be used to detectany analyte which has heretofore been assayed using known immunoassayprocedures, or is detectable by such procedures, using polyclonal ormonoclonal antibodies or other proteins. Various specific assayprotocols, reagents, and analytes useful in the practice of theinvention are known per se, see, e.g., U.S. Pat. Nos. 4,313,734, and4,366,241.

The combination of features believed to be responsible for the excellentsensitivity and reproducibility of assays constructed in accordance withthe invention is the use of the novel biphasic chromatographic substrateand the use of a metal sol or other colored particle as a marker systemwhich permits direct visual observation of color development. Afiltration means which limits the introduction to the test site ofcontaminants from the sample also may be included.

The assay is conducted by simply placing the inlet of the device incontact with a liquid test sample. The casing of the device may beconfigured to permit direct contact with a body fluid, or as a dipstickfor dipping in a container of body fluid or other test solution. Aftercontact with the test fluid, one then merely waits for the test sampleto pass through the biphasic chromatographic substrate and into reactivecontact with the test site (and optionally one or more control sites)visible through a window or windows in the device's exterior casing. Ina preferred embodiment, the labeled binding member specific for theanalyte is disposed in preserved form on the release medium in the flowpath within the device. If analyte is present in the sample, it passesthrough the inlet and the interior of the device along thechromatographic substrate where, in the sandwich embodiment, it reactswith labeled binding protein, and a capturable component conjugated withan affinity agent. The complex formed by the analyte, labeled bindingmember and the affinity conjugate then reacts with a capture affinityagent immobilized at the capture site which is specific for the affinityagent on the conjugate. A complex forms at the capture site comprisingimmobilized capture agent-capturable conjugate-analyte-labeled bindingmember. The presence of the complex, and thus the analyte, is indicatedby the development of color caused by aggregation of the metal solparticles at the capture site.

From the foregoing, it will be apparent that the success of the testprocedure is dependent on analyte present in the sample reacting withthe labeled binding member, or on reproducible competition between theanalyte and the binding member for sites of attachment at the capturesite. In accordance with the invention, as noted above, the labeledbinding member and capturable conjugate preferably are disposed inpreserved form, e.g., air dried or freeze-dried, on the release mediumwithin the device upstream of the capture and control sites. Analyte, ifany, passing up through the device and entrained within the liquid movesinto contact with the labeled binding member and capturable componentforming an immune complex or initiating competition in situ as flowcontinues, which complex ultimately is captured by reagents immobilizedon the capture medium.

Referring now to the drawings, FIGS. 1A–E illustrate schematically anembodiment of a test device 5 constructed in accordance with theinvention useful in explaining its principles of construction. Itcomprises an outer, molded casing 10 which defines a hollow, elongateenclosure. Casing 10 defines a test liquid inlet 14 and an opening 16comprising a window through which the capture and control sites arevisible. As illustrated in FIGS. 1A–E window 16 is disposed on a side ofthe casing 10 opposite sample inlet 14. This configuration reduces theincidence of contamination of the test site which is disposed in theinterior of casing 10 and is exposed through window 16. Casing 10further defines vent openings 38, 40 and 42 located along the sides andat the distal end of casing 10. Vent opening 38 reduces the incidence of“vapor lock” within the device during use. The presence of openings 40and 42 help to reduce “flooding” of the chromatographic substrate, whichmay occur when the user applies too much sample to the device.

FIG. 2 illustrates schematically a preferred embodiment of the assaymaterials, which when the device is fully assembled, are disposed insidecasing 10. The assay materials comprise absorbent material 12, biphasicchromatographic substrate 18 and reservoir material 24. The assaymaterials and the interior of casing 10 together define a flow pathpassing generally from right to left in FIGS. 1A, B and C. When the testdevice is placed with inlet 14 disposed within or otherwise in contactwith a liquid sample, the liquid is transported by capillary action,wicking, or simple wetting along the flow path downstream throughabsorbent 12, along chromatographic substrate 18, and into reservoir 24,generally as depicted by the arrow. Absorbent material 12 disposedinwardly of the inlet 14 also serves as a filter which can remove fromimpure test samples particulate matter and interfering factors.

FIG. 3 illustrates schematically the biphasic chromatographic substrate18, comprising a release medium 30 and a capture medium 32. Releasablydisposed on release medium 30 is a band 26 of dehydrated labeled bindingmember, e.g., antibody-metal sol. As the liquid sample moves past band26, the labeled binding member becomes entrained in the liquid,reconstituted, and reacts or competes with any analyte present in theliquid sample. Disposed downstream of the labeled binding member is aband 28 of dehydrated capturable complex. The capturable complexcomprises a binding member which binds to a second epitope of theanalyte, e.g. an antibody, and a capturable affinity component, e.g.,biotin. The capturable complex also becomes entrained in the liquidsample as it advances along substrate 18.

Immobilized on capture medium 32 are, respectively, capture site 34 andcontrol site 36. In FIG. 3, the control and capture sites areillustrated as being disposed serially along the flow path.Alternatively, the control and capture site or sites may be disposedside by side or in other spacial relationships. Capture site 34comprises a preselected quantity of a capture affinity member specificfor the capturable affinity component disposed on the release medium.The capturable component is immobilized in place within the flow path.For example, when the capturable affinity member is biotin, the capturecomponent may be streptavidin. Control site 36 comprises immobilizedantisera or antibody specific for the labeled binding member.

FIG. 4 illustrates schematically a side view of the operative portion ofthe assay materials. As shown, absorbent material 12 is disposedproximate release medium 30, and overlaps release medium 30 at one end.Release medium 30 in turn overlaps capture medium 32, which is disposeddistal to release medium 30. Reservoir 24 overlaps the distal end ofcapture medium 32. These four components together form a single fluidpath, and cooperate to cause sample liquid to flow from absorbent 12along release medium 30 and capture medium 32 into reservoir 24.

FIG. 5 illustrates schematically a currently preferred embodiment of theoperative portion of the assay device. As shown, release medium 30 hasreleasably disposed thereon a band of labeled binding member 26, whichin the preferred embodiment is a monoclonal antibody conjugated to goldsol particles. Disposed downstream from band 26 is band 28 comprisingthe capturable component, which in the preferred embodiment is a secondmonoclonal antibody specific for the same analyte conjugated to biotin.Band 28 also is releasably disposed on release medium 30. Locateddownstream of release medium 30 is capture medium 32 having immobilizedthereon capture site 34, which in the preferred embodiment isstreptavidin. Located on capture medium 32 downstream of capture site 34is control site 36, which in the preferred embodiment is polyclonalantisera specific for the labeled antibody of band 26.

The invention is not limited by the precise nature of the capture site34 and corresponding control site 36, and in fact, control site 36 maybe entirely eliminated if desired. Generally, antibody or other affinityagent can be immobilized at capture site 34 and control site 36 usingabsorption, adsorption, or ionic or covalent coupling, in accordancewith methods known per se. Capture medium 32 preferably is selected tobind the capture reagents without the need for chemical coupling.Nitrocellulose and nylon both permit non-chemical binding of the capturecomponent and control reagent.

As shown in FIG. 5, disposed downstream of capture medium 32 isreservoir 24 comprising a relatively large mass of absorbent orsuperabsorbent material. The purpose of reservoir 24 is to ensure that areasonably large amount of test liquid is drawn across thechromatographic medium.

FIG. 6 is a schematic illustration of an embodiment of the assaymaterials useful in performing dipstick assays. In the embodiment shown,sample absorbent 12 is omitted, and release medium 30 acts as the sampleabsorbent.

Polyclonal antisera and monoclonal antibodies or fractions thereofhaving specific binding properties and high affinity for virtually anyantigenic substance which are useful in the present invention as bindingmembers and capture materials are known and commercially available, orcan be produced from stable cell lines using well known cell fusion andscreening techniques. The literature is replete with protocols forproducing and immobilizing proteins. See, for example, LaboratoryTechniques in Biochemistry and Molecular Biology, Tijssen, Vol. 15,Practice and Theory of Enzyme immunoassays, chapter 13, TheImmobilization of Immunoreactants on Solid Phases, pp. 297–328, and thereferences cited therein.

Metal sols and other types of colored particles useful as markersubstances in immunoassay procedures are also known per se. See, forexample, U.S. Pat. No. 4,313,734. For details and engineering principlesinvolved in the synthesis of colored particle conjugates seeHorisberger, Evaluation of Colloidal Gold as a Cytochromic Marker forTransmission and Scanning Electron Microscopy, Biol. Cellulaire, 36,253–258 (1979); Leuvering et al., “Sol Particle Immunoassay”, J.Immunoassay, 1 (1): 77–91 (1980), and Frens, “Controlled Nucleation forthe Regulation of the Particle Size in Monodisperse Gold Suspensions”,Nature, Physical Science, 241: 20–22 (1973).

In one currently preferred embodiment, the immunoassay device of thepresent invention is designed to detect human pregnancy. In thisembodiment, the labeled binding member is a monoclonal antibody (MAb)against human chorionic gonadotropin (hCG) labeled with colloidal gold.For this purpose, MAb designated 2G9 (available from Carter-Wallace,Inc.) is preferred. Anti-hCG antibodies labeled with biotin are used forthe capturable complex. Monoclonal antibodies which can be used for thispurpose include the hCG specific monoclonal antibodies designated 2B2and B109 (available from Carter-Wallace, Inc.) and CCF01 (available fromScripps Laboratory). Methods for conjugating biotin to antibodies arewell-known and do not form a part of the present invention. In thepresent preferred embodiment, the capture site comprises streptavidin,which has a high affinity for biotin. A control site preferably islocated downstream of the capture site. The control site has immobilizedthereon goat anti-mouse IgG specific for the labeled anti-hCG (availablefrom Scantibodies Laboratory).

In another preferred embodiment the present immunoassay device isdesigned to detect human ovulation. In this embodiment, the labeledbinding member comprises MAb 2G9, which is specific for luteinizinghormone (LH) and hCG, labeled with colloidal gold. The capturablecomplex comprises biotinylated LH-specific MAb LH26 (available fromCarter-Wallace, Inc.). The capture site preferably comprisesstreptavidin and the control site comprises goat anti-mouse IgG specificfor the labeled MAb.

In another embodiment, the device may be adapted to detect infectiousagents, such as streptococcus. In this embodiment, the labeled bindingmember is a rabbit polyclonal antibody specific for streptococcuslabeled with colloidal gold or other direct marker. The capturablecomplex is the same polyclonal antibody conjugated to biotin, and thecapture and control components comprise streptavidin and goatanti-rabbit IgG.

The casing 10, can take various forms. It will typically comprise anelongate casing comprising interfitting parts made of a plastic materialsuch as polyvinyl chloride, polypropylene, polystyrene or polyethylene.Its interior flow path will contain a relatively inert material or acombination of materials suitable for facilitating transport of theliquid. Casing 10 may be adapted for direct contact with a sampleliquid, as shown in the embodiment illustrated in FIGS. lA–E, or may beadapted to dipstick form, which is not shown herein, but is well knownin the art. A currently preferred design for casing 10 is described inU.S. Pat. No. Des. 361,842 and in U.S. Pat. No. 5,739,041.

From the foregoing it should be apparent that the advantages inreproducibility, sensitivity, and avoidance of false positives of assaysystems constructed in accordance with the invention are traceable to acombination of features of the invention. In use, the biphasicchromatographic substrate results in efficient transport of the reagentswhich allows more sensitive detection of analyte.

The present invention will now be further particularly described withreference to the following Exemplification. In the Exemplification, thetest devices are described with reference to FIGS. 1–6 of theaccompanying drawings which have been briefly described hereinabove.

EXEMPLIFICATION

Pregnancy Test

The currently preferred configuration for the casing for the test deviceembodying the invention is shown in FIGS. 1A–E. The currently preferredconfiguration of the test materials including sample absorbent, biphasicchromatographic substrate and reservoir is shown in FIGS. 2–5. Amodification of the test materials depicted in FIGS. 2–5 is shown inFIG. 6.

As shown in FIGS. 1A–E, the preferred test cell of the inventioncomprises a pair of interfitting polymeric parts which, when the deviceis assembled, define an enclosure. Casing 10 is designed to receive thebiphasic chromatographic substrate and related absorbents shown in FIGS.2–5. The assay materials are disposed within casing 10 such that theclear polymer backing 46 is disposed opposite window 16 (FIG. 1A). Theresults of the assay can be read through the clear polymer layer 46, andthe presence of layer 46 prevents contamination of the test site duringuse. In operation, test liquid applied through inlet 14 is absorbed bysample absorbent 12, and soaks along chromatographic substrate 18 whichdefines the flow path, into reservoir volume 24. In the dipstickembodiment, the chromatographic substrate shown in FIG. 6, which lacksabsorbent material 12, is disposed in a casing adapted to receive it,which is shown in FIG. 7.

In the currently most preferred embodiment, the absorbent 12 is bondedhydrophilic polyester (American Filtrona); the release medium is eitherS&S 903 paper or S&S GB002 paper (both from Schleicher & Schuell); thecapture medium is nitrocellulose membrane cast (or laminated) onto clearpolyethylene terephthalate (PET) (available from Millipore Corp.); thereservoir 24 is S&S 300 paper (Schleicher & Schuell). The release andcapture media were laminated onto 5 mil clear PET precoated with anadhesive (available from Adhesives Research). The dimensions ofabsorbent material 12 are approximately 5.0×1.27×0.25 cm (2.0×0.5×0.1inches) on each side. The dimensions of release medium 32 of thebiphasic chromatographic substrate 18 are approximately 2.8×0.8×0.06 cm(1.1×0.32×0.025 inches), and for the capture medium, approximately2.5×0.8×0.018 cm (1×0.32×0.007 inches). The dimensions of the reservoirabsorbent pad are approximately 2.0×0.26×1.06 cm (0.8×0.1×0.4 inches). Anumber of these substrates were produced and further treated to adaptthem to detect pregnancy by assay of urine for the presence of humanchorionic gonadotropin (hCG). The test reagents were hCG monoclonalantibody 2G9 (obtained from Carter-Wallace, Inc.) labeled with colloidalgold 15–30 nm in size; biotinylated hCG specific monoclonal antibodyCCF01 (obtained from Scripps Laboratory); streptavidin, lyophilizedmaterial reconstituted in phosphate buffer to a concentration of 2mg/ml; and goat anti-mouse IgG, solution adjusted to a concentration of1 mg/ml in phosphate buffer. The test reagents were positioned on themedia as shown in FIGS. 3 and 5.

Test Protocol

Two samples of the pregnancy assay were prepared using biphasicchromatographic media as described above. One sample designated “Test 1”was prepared using as the capture medium a nitrocellulose membrane whichhad been laminated onto a polyester film. The other sample, designated“Test 2” was prepared using as the capture medium a nitrocellularmembrane which had been cast onto a polyester backing. All other aspectsof the biphasic chromatographic media were identical.

The test was carried out by applying solutions containing knownquantities of commercially available hCG to the proximal end of thechromatographic media, and permitting the test solutions to proceedthrough capillary action through the biphasic medium.

For comparison, a commercially available pregnancy test kit (FirstResponse™, Carter-Wallace, Inc., New York, N.Y.) was subjected to thesame procedure. The commercial test kit uses a single phasechromatographic medium consisting of a hydrophilic cellulosic material.The differences between the test samples and the commercial test areshown in Table 1.

TABLE 1 Commercial Test Product Samples Chromatographic Monophasic-Bi-phasic-comprising a Medium consisting of capture medium (nitro-hydrophilic cellulose membrane) cellulosic and a release medium material(hydrophilic cellulosic material) Reagents Mobile reagents- MobileReagents- (Bottom to top) 1. Labeled anti- 1. Labeled antibody hCGantibody 2. Biotinylated antibody 2. Biotinylated Capture reagents-antibody 3. Streptavidin Capture reagents- 4. Goat anti-mouse IgG 3.Streptavidin coupled to latex 4. Goat anti-mouse IgG coupled to latexMode of Via latex beads Passive adsorption Immobilization of ontohydrophilic onto nitrocellulose Capture Reagents cellulosic membranematerial Mobile Reagents Direct application Direct application onto ontothe hydrophilic the hydrophilic cellulosic cellulosic material materialUpstream Blotting paper Same Reservoir made of a blend of cotton linterfibers Chromatographic Mono-phasic Bi-phasic Component Sample Two 50 mm× 12.5 mm × 5 mm One 50 mm × 11.4 mm × 2.5 mm Absorbent cellulosehydrophilic acetate pads polyester materialThe results of the test procedure are shown in Table 2:

TABLE 2 Comparison of Reaction Times for Commercial and ImprovedPregnancy Tests Test 1 Test 2 Using Using Mylar hCG Level CommercialLaminated Membrane Cast Membrane (mIU) Rxn/time Rxn/time Rxn/time 0neg/1:45 neg/1:35 neg/1:30 25 neg/2:06 pos/1:15 neg/1:45 50 neg/1:45pos/0:50 pos/0:50 100 pos/2:00 pos/0:40 pos/0:30 300 pos/2:00 pos/0:40pos/0:35

The pink color was clearly visible at 50 mIU of human chorionicgonadotropin for both test samples, but not for the commercial product.The results indicate that the test of the present invention can detectpregnancy in a human as early as the day of a missed menstrual period.In initial stages of testing, approximately 50 negative samples fromvarious sources had been run with no false positives or even border-linecases. In contrast, at 50 mIU of hCG, the commercial pregnancy testshowed a negative result.

Table 3 illustrates the amounts of reagents needed for the pregnancytest (Test 2) made using the biphasic substrate of the present inventioncompared to the commercial test. As shown in Table 3, tests of thepresent invention require significantly less reagent and (as shown inTable 2) are both more sensitive and more accurate.

TABLE 3 Comparison of Reagent Usage TEST 2: PERCENT REAGENT COMMERCIALIMPROVED DECREASE Gold labeled 0.45 OD₅₃₃ 0.048 OD₅₃₃ 89 antibodyBiotinylated   1 ug 0.1 ug 90 antibody Streptavidin 7.8 ug 0.8 ug 90Goat anti-mouse 7.5 ug 0.8 ug 89 lgG

Table 4 shows the results of actual tests carried out using the presentpregancy test device. All versions listed in Table 4 contain thebiphasic medium and reagents described above, but differ in theconfiguration of casing 10. It was found, surprisingly, that certainphysical modifications to casing 10, particularly the introduction ofvents 38, 40 and 42 (FIGS. 1B, 1E), resulted in significant higheraccuracy and fewer invalid results. The differences between thedifferent versions shown in Table 4 is as follows:

Version 1

This version had long posts that were easily broken when the componentswere stored in plastic bags. The devices easily came part when droppedfrom a desk top.

Version 2

In this version the posts were shortened to eliminate breakage. A urinestop was added to prevent urine from bypassing the release medium andwetting the membrane.

Version 3

This version added posts to prevent the device from coming apart whendropped. Additional urine stops were included to further preventflooding of the membrane. A crossbar was added to the top housing thepush the upstream absorbent against the membrane to assist in sampleflow and gold clearance.Version 4Version 3 devices were modified by manually cutting vents in the device.Two long side vents and one short base vent were cut in the top housingto prevent membrane flooding.Modified Version 4Version 4 devices were modified by cutting a window in the top housingsimilar to the urine collection area on the bottom housing. This devicewas called “two-sided”—sample could be applied to either the front orback of the device.

Version 5 represents the currently preferred embodiment of the pregnancytest device. As shown in Table 4, the tests were all completed in lessthan five minutes, with no invalid results.

EQUIVALENTS

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications to adapt it to various usages and conditions. Suchembodiments are intended to be included within the scope of thefollowing claims.

1. A device for determining the presence in a liquid sample of ananalyte wherein liquid sample deposited on a proximal end of the devicetravels downstream along a liquid path to a distal end of the device,the device comprising: a biphasic substrate comprising (1) a releasemedium of a first material and, in fluid communication and downstreamthereof, (2) a capture medium of a microporous, second, differentmaterial, wherein located at the release medium of the biphasicsubstrate for release therefrom is (i) a labeled conjugate comprising abinding member reactive with a first epitope of the analyte labeled witha detectable marker, and (ii) a capturable component reactive with asecond epitope of the analyte, such that if the analyte is present inthe sample, the analyte produces a complex comprising the labeledconjugate, the analyte and the capturable component, and located at thecapture medium of the biphasic substrate is a capture site for capturingthe complex, the capture site having immobilized thereon a capturecomponent having a binding affinity for the capturable component.
 2. Thedevice of claim 1 further comprising a control site located downstreamof the capture site, the control site having immobilized thereon anagent having a binding affinity for the labeled conjugate.
 3. The deviceof claim 1 wherein the capture medium is laminated to or cast on atransparent polymeric material.
 4. The device of claim 1 comprising asample absorbent located upstream of the biphasic substrate at theproximal end of the device.
 5. The device of claim 1 further comprisinga residual absorbent located downstream of the biphasic substrate at thedistal end of the device.
 6. The device of claim 1 wherein the bindingmember of the labeled conjugate is an antibody.
 7. The device of claim 1wherein the capturable component is a biotinylated antibody.
 8. Thedevice of claim 7 wherein the immobilized capture component comprisesavidin, streptavidin or an antibiotin antibody.
 9. The device of claim 1wherein the immobilized capture component comprises avidin,streptavidin, or an antibiotin antibody.
 10. The device of claim 1wherein the second material of the biphasic substrate is nitrocelluloseor nylon.
 11. The device of claim 1 wherein the first material of thebiphasic substrate is absorbent paper.
 12. The device of claim 1 whereinthe release medium and the capture medium of the biphasic substrate areboth immobilized on a single backing support.
 13. A device fordetermining the presence in a liquid sample of an analyte wherein liquidsample deposited on a proximal end of the device travels downstreamalong a liquid path to a distal end of the device, the devicecomprising: a biphasic substrate comprising (1) a release medium of afirst material and in fluid communication and downstream thereof, (2) acapture medium of a second, different material of nylon ornitrocellulose, wherein the release medium and the capture medium areboth immobilized on a single backing support, wherein located at therelease medium of the biphasic substrate for release therefrom is (i) alabeled conjugate comprising a first antibody that binds a first epitopeof the analyte labeled with a detectable marker, and (ii) a capturablecomponent comprising a biotinylated second antibody that binds a second,different epitope of the analyte, such that if the analyte is present inthe sample, the analyte produces a complex comprising the labeledconjugate, the analyte and the capturable component, and located at thecapture medium of the biphasic substrate is a capture site for capturingthe complex, the capture site having a capture component immobilizedthereon for biding the capturable component.
 14. The device of claim 13further comprising a control site located downstream of the capturesite, the control site having immobilized thereon an agent havingbinding affinity for the labeled conjugate.
 15. The device of claim 13,wherein the capture medium is laminated to or cast on a transparentpolymeric material.
 16. The device of claim 13 further comprising asample absorbent located upstream of the biphasic substrate at theproximal end of the device.
 17. The device of claim 13 furthercomprising a residual absorbent located downstream of the biphasicsubstrate at the distal end of the device.
 18. The device of claim 13,wherein the first material of the biphasic substrate is absorbent paper.19. The device of claim 13, wherein the capture component is avidin orstreptavidin.